Flexanalysis software

After tryptic in gel-digestion, overnight at 37°C [24] (link), the differentially expressed spots were excised from preparative gels and analyzed by mass spectrometry (MS) to identify amino acid sequences, using a Bruker Ultraflex III (Bruker, Bremen, Germany) operating in reflectron mode. This instrument was equipped with a Nd:YAG smartbeam laser to acquire positive-ion MALDI mass spectra over a mass range of m/z 800–4000. Spectral processing and peak list generation were implemented by Bruker flexAnalysis software (version 3.3, Bruker Daltonics) for MS and MS/MS spectra. For each protein spot, the most intense precursor ion signals in each MS spectrum were analyzed by MS/MS fragmentation in LIFT mode. α-cyano-4-hydroxycinnamic acid was used as matrix. Spot identifications were performed by querying the Mascot database. Trypsin cut, carbamidomethyl (C) as fixed, oxidation (M) as variable, a maximum of one missed cleavage allowed, were imposed as modifications in the search parameters. Peptide tolerance and MS/MS tolerance were set at 250 ppm and 0.5 Da respectively.

For mass spectrometry analysis of products, 2 μL of a 9 mg/mL mixture of 2,5-dihydroxybenzoic acid (DHB) in 30% acetonitrile was applied to a MTP 384 ground steel target plate TF (Bruker Daltonics GmbH, Bremen, Germany). One-microliter sample was then mixed into the DHB droplet and dried under a stream of air. The samples were analysed with an Ultraflex2 MALDI-ToF/ToF instrument (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Nitrogen 337-nm laser beam. The instrument was operated in positive acquisition mode and controlled by the FlexControl 3.3 software package. All spectra were obtained using the reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26, and pulsed ion extraction of 40 ns in the positive ion mode. The acquisition range used was from m/z 300 to 3,000. The data was collected from averaging 400 laser shots, with the lowest laser energy necessary to obtain sufficient signal-to-noise ratios. Peak lists were generated from the MS spectra using Bruker FlexAnalysis software (Bruker Daltonics GmbH, Bremen, Germany) (Version 3.3).

The MALDI MS data were acquired and processed in FlexAnalysis software (Bruker) and m/z values, intensities, peak area and other data were exported as Excel files. The peak area of each glycan was the addition of both fully permethylated and underpermethylated glycan peaks (14 Da lower than the fully), which was then normalized to the sum of all glycan peak areas and represented as a percentage of the total peak area. Analysis of branching degree and fucosylation degree was conducted by Excel and visualized with Prism 7 (La Jolla, CA). Summary statistics are used to describe the patient characteristics. Discrete variables such as gender are summarized using frequency tables and continuous variables are summarized using median, mean and standard deviation. Boxplots are used to illustrate the marker distributions. Wilcoxon test or Kruskal-Wallis test are used to compare the value of continuous variables across groups.
Logistic regression model is used to combine trifucosylation of AGP with AFP and other labs in the marker panel development. Receiver operation characteristic (ROC) curves are constructed. The area under the curve (AUC) is calculated, and its 95% confidence interval (CI) is estimated using the bootstrapping method. All the analyses are performed using R statistical software (https://cran.r-project.org).

The matrix trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile was dissolved at a final concentration of 30 mg/mL in 90% v/v aqueous acetonitrile solution with 0.1% formic acid. A loop of the bacterial strain was smeared onto the stainless steel 384 target plate and 2 μL matrix solution was applied on top of the sample. The samples were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS) analysis on a Bruker Autoflex Speed mass spectrometer in reflector/positive mode. Each sample was ablated 1000 times within 1 s with a laser energy output of 70%. A mass-to-charge (m/z) window of 500–3000 was applied to monitor the production of lipopeptides. All mass data were analyzed using the FlexAnalysis software (V 3.4, Bruker Daltonik GmbH, Bremen, Germany).

PITCR-associated peptides were dissolved in 1 mM sodium phosphate buffer (pH 7.4, filtered). The matrix α-cyano-4-hydroxycinnamic acid (TCI C1768) was dissolved in 75% HPLC-level acetonitrile coupled with 0.1% TFA and sonicated 15 min, RT. The dissolved samples were mixed with the dissolved matrix. After that, the mixed matrix samples were loaded onto the MSP target plate (Bruker, Billerica, MA, USA) drop by drop and dried using filtered air. The Bruker Microflex MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA) was calibrated with ProteoMass MALDI-MS calibration standards (Sigma-Aldrich I6279-5X1VL, I6154-5X1VL, C8857-5X1VL, and P2613-5X1VL). All PITCR-associated peptides were measured in a negative mode. The pHLIP was measured in a positive mode. Data were analyzed using FlexAnalysis software (Bruker, Billerica, MA, USA) and graphs were plotted using Origin 9.1 (research lab) software.